This proposal will focus on an experimental model of inflammatory bowel disease, i.e., the C3H/HeJBir mouse, which is a high susceptibility phenotype that can develop colitis spontaneously under certain environmental conditions. This strain was developed in previous cycles of this Program Project. We have recently shown that CD4+ T cells from such mice are reactive to protein antigens of enteric bacteria but not to food or epithelial cell antigens. Such bacteria-reactive T cells can transfer colitis to immunodeficient recipients. Such bacteria-reactive T cell lines of defined cytokine phenotype in vitro, including Th1, Th2, Th0, and TR1. These T cell lines will be used in adoptive transfer experiments to understand their effects in vivo. The aim of this project is a greater understanding of the enteric bacterial antigens, the regulator and effector cells that recognize them, and the T cell receptors used in such recognition in both normal and inflamed colon. The hypothesis is that murine colitis is mediated by a restricted set of pathogenic CD4+ effector T cells responding to antigens of the enteric bacterial flora, antigens to which the host is normally tolerant, that in addition to antigen-T cell receptor interactions, co-stimulatory molecules such as CD40L and CD28, as well as IL-12, play a critical role in activation and expansion of pathogenic T cells; and that progression to colitis is due at least in part to an imbalance between effector and regulatory T cell subsets in the intestinal mucosa. To this end, four specific aims will be addressed. 1) The molecular pathogenesis of colitis after transfer of enteric-bacterial reactive CD4+Bir T cell lines into C3H-SCID recipients will be defined, including the localization and expansion of such cells after transfer, the cytokines that are expressed in the mucosa, the co-stimulatory molecules that are required, and the role of IL-12. 2) Whether IL-10 producing T regulatory 1 (Tr1) cells prevent or treat colitis and whether they maintain normal intestinal homeostasis. These studies will focus particularly on a enteric bacterial-reactive Tr1 cell line that has been established in vitro in the laboratory.3) We will ask whether there is a state of immologic tolerance to antigens of the enteric bacterial flora in normal mice and whether this is impaired in colitic mice. To this end, defined recombinant intestinal bacterial antigens have been cloned from the bacterial flora. 4) We will a sk whether the T cell repertoire of intestinal CD4+ T cells is skewed in colitic mice. These studies will add significantly to our understanding of the interactions between bacterial antigens and mucosal T cells. The long-term goal of this project is development of novel forms of therapy for patients with colitis based on modulation of such interactions.